The overall goal of these studies is to improve the treatment of non-muscle invasive bladder cancer by identifying second line agents that are effective in the scenario of BCG resistance and hopefully for all nonmuscle invasive bladder cancer in future years. During the course of the last cycle of SPORE funding, we provided strong evidence for effective gene transfer and potential clinical efficacy when intravesical adenoviral-mediated interferona(IFNa) gene therapy was administered with Syn3 (Ad-IFNa/Syn3). Approximately 50% of the treated patients, all of which were BCG resistant, obtained a complete remission (CR). We also found that Ad-IFNa induces cancer cell death in human bladder cancer cells that are insensitive to the recombinant IFNa protein itself, and that cell death occurs via direct and indirect (bystander) mechanisms. Moreover, normal urothelial cells appear to be resistant to Ad-IFNa. In this renewal we plan to complete a Phase IB study of intravesical Ad-IFNa/Syn3 to optimize the schedule by determining whether therapy on Day 1 and an additional instillation on Day 4 will enhance gene transfer and result in higher and more sustained urine IFNa levels than were achieved with a single treatment In patients achieving a CR at 90 days we will repeat treatment on Day 1 and 4 to determine whether redosing with this schedule will provide for effective gene transfer. TRAIL, M30 and M65 levels will also be measured in the urine as possible markers of clinical efficacy and tumor cell death. Subsequently we plan to participate in and lead a multicenter Phase II trial based upon the results of Phase IB study. The isolation and identification of the soluble factor(s) responsible for the Ad-IFNa produced bystander cell death on cancer cells will also be a focus. In addition to its obvious value as a biomarker of Ad-IFN activity, the bystander factor(s) could itself have clinical utility. Various methods will be utilized in these studies, including size exclusion concentration, size-exclusion FPLC column subfractionation and mass spectroscopy.